The preparation and administration of many drugs and therapeutic proteins following freeze-drying (lyophilization) is known. Advances in polymer chemistry and biomaterials have achieved the delivery of drugs via attachment to graft materials. For example, heparin and urokinase bonding to vascular graft materials has been used in an attempt to improve long-term patency of vascular grafts. Chemotherapy drugs have been impregnated into wafers for time-release delivery. None of these systems, however, deliver DNA with the goal of persistent therapeutic protein expression in the host cells.
Known viral and non-viral (e.g. lipid-mediated, electroporation-enhanced) gene therapy delivery systems rely on the delivery of gene vectors in solutions. The solutions may be injected intravenously, intramuscularly, subcutaneously, or may even be administered orally. These approaches are hampered by the fact that many gene therapy vectors or formulations are not stable (i.e., they lose the ability to infect cells and deliver DNA) when the vector is exposed to alterations in conditions like temperature, hydration, or pH. In addition, these routes may lead to undesirably rapid biodistribution of the delivered virus to organs distant from the tissue of delivery (e.g., within seconds after intravenous delivery, or within minutes to hours after intramuscular delivery). Accordingly, a need exists for methods in which gene delivery may be accomplished in a time-controlled or delayed manner, and potentially with delayed biodistribution of virus distant from the target tissue.
Adeno-associated virus (AAV) is a nonpathogenic, helper-dependent member of the parvovirus family. One of the identifying characteristics of this group is the encapsidation of a single-stranded DNA (ssDNA) genome. The separate plus or minus polarity strands are packaged with equal frequency, and either is infectious. At each end of the ssDNA genome, a palindromic terminal repeat (TR) structure base-pairs upon itself into a hairpin configuration. This serves as a primer for cellular DNA polymerase to synthesize the complementary strand after uncoating in the host cell. Adeno-associated virus generally requires a helper virus for a productive infection. Although adenovirus (Ad) usually serves this purpose, treatment of AAV infected cells with UV irradiation or hydroxyurea (HU) will also allow limited replication.
Recombinant AAV (rAAV) gene delivery vectors also package ssDNA of plus or minus polarity, and must rely on cellular replication factors for synthesis of the complementary strand. While it was initially expected that this step would be carried out spontaneously, by cellular DNA replication or repair pathways, this does not appear to be the case. Early work with rAAV vectors revealed that the ability to score marker gene expression was dramatically enhanced when cells were co-infected with adenovirus, or transiently pretreated with genotoxic agents. Similar induction of rAAV vectors has been observed in vivo following treatment with Ad, ionizing radiation, or topoisomerase inhibitors. However, the effect was highly variable between different tissues and cell types.
The effort to establish the efficiency of AAV-mediated gene delivery following complete desiccation was described by Rabinowitz et al. in May, 1998 at the Third International Cancer Gene Therapy Meeting. At that time, it was shown that recombinant AAV remains infectious and competent for transduction (gene delivery leading to expression of the transgenic protein) in a broad range of temperatures, pH, and hydration states. Since that time, the present inventors have performed additional studies of AAV-mediated gene delivery following desiccation of the virus, based upon the reasoning that one limitation of gene therapy with viral vectors delivered to the bloodstream, or even delivered locally to tissues like the lung or muscle in large doses, is virus scatter to many sites beyond the intended site of action.
One disadvantage of virus scatter is that generally, too little of the therapeutic gene is present or delivered to the site where it is intended. For example, a CFTR gene intended to be delivered to the lung as a treatment for cystic fibrosis will have decreased utility if most of the virus goes to the liver and spleen. Another problem with known gene delivery systems is that it is difficult to judge an accurate dosage of the gene vector required to achieve the desired therapeutic effect. Finally, if the viral vector has potentially toxic effects upon exposure to organs outside of the target, it is desirable to localize the therapeutic treatment to the target only and decrease viral scatter. A persistent concern with gene therapy is the theoretical possibility of unwanted gene delivery vector spreading to subjects' gonads, thus perhaps leading to insertional mutagenesis to germline cells. Accordingly, precise direction and amount of delivery of gene therapy vectors continues to be a priority in the development of safe and reliable gene delivery systems.